SERPINE1 Gene Is a Reliable Molecular Marker for the Early Diagnosis of Aortic Dissection

With the acceleration of population aging, the detection rate of aortic dissection has increased. The incidence rate of aortic dissection has increased year by year and has become a serious threat to human health. However, the current clinical treatment of aortic dissection is mainly limited to surgery (including intracavity), but the complexity of the disease and the high risk of surgery seriously affect the overall treatment effect of the disease. Therefore, an in-depth study of the pathogenesis of aortic dissection and the development of early diagnosis methods is not only expected to control the development of aortic dissection but also to improve the existing clinical treatment effect. Based on the bioinformatics analysis of the related mRNA sequence data of aortic dissection in GEO database, the gene expression regulatory network of aortic dissection was constructed. Through the screening of key node genes, the key factors (molecular markers) that may affect the occurrence of aortic dissection were obtained, and their functions were tested in human aortic smooth muscle cells (HAoSMC). Finally, it was concluded that SERPINE1 gene is a reliable molecular marker for the early diagnosis of aortic dissection.


Introduction
Aortic dissection (AD) is a fatal vascular disease in which blood enters the middle layer of the vessel wall through a tear in the inner lining of the aortic wall and forms a dissecting hematoma along the aortic wall [1]. e incidence of aortic coarctation is 0.005% to 0.03% per year [2,3]. It is common, but the mortality rate is high. e mortality rate increases by 1% per hour for the first 48 hours and a large number of patients die en route to the hospital or before the diagnosis is confirmed. Current treatment options include percutaneous endovascular exclusion and open-heart surgery. ese treatments are not only very traumatic for the patient but also put more financial pressure on the patient and his family. According to the patient's blood pressure, tumor diameter, and other comprehensive consideration, surgical treatment is worth considering. Because of myocardial infarction, renal failure, stroke, paraplegia, and other postoperative complications, even patients undergoing surgery have a higher risk of death. Moreover, with the acceleration of population aging, the detection rate of aortic dissection has increased and the incidence rate of aortic dissection has increased year by year and has become a serious threat to human health. However, clinical management of aortic coarctation is currently limited mainly to surgery (including endoluminal), but the complexity of the disease and the high risk of surgery seriously affect the overall outcome of the disease. Some studies have shown that the mortality rate in the early postoperative period after aortic coarctation is as high as 9%-30% [4]. erefore, an in-depth study of the pathogenesis of aortic coarctation and exploration of early diagnostic methods is expected not only to control the development of aortic coarctation but also to further improve the existing clinical treatment outcomes.
At present, the known causes of aortic dissection include congenital genetic variation, connective tissue lesions, infection, trauma, and other factors [5]. Several well-studied signaling pathways in AD development include TGF-β signaling pathway, angiotensin II signaling pathway, and focal adhesion, and actin cytoskeleton regulation [6][7][8].
ere are also regulatory mechanisms regarding the contractile function of vascular smooth muscle, such as the ACTA2 gene encoding ɑ actin and the MYH11 gene encoding β myosin heavy chain, which are important components of actin and myosin, respectively, whereas the contraction of smooth muscle is produced under the premise of cross-linking of actin and myosin; therefore, the abnormal expression of ACTA2 and MYH11 genes can affect the contractile function of vascular smooth muscles [9,10]. Extracellular matrix (ECM) is the main component that forms the morphology of the aortic vessel wall, but in the vascular wall tissue of patients with thoracic aortic dissection, this component has obvious abnormalities [11]. Collagen and elastin play an important role in maintaining the elastic properties of the aortic wall [12]. Although a large number of studies have shown that typical pathological changes of aortic dissection and matrix metalloproteinases (MMPs) play a direct role in the degradation of aortic extracellular matrix [13], the interaction mode of these factors and their upstream regulatory factors are still unclear. e aim of this study was to construct a regulatory network for aortic coarctation gene expression by screening key node genes and key factors (molecular markers) that may influence their occurrence, through bioinformatic analysis of aortic coarctation mRNA sequence data in the GEO database. Aortic coarctation was obtained and their functions were verified in human aortic smooth muscle cells (HAoSMCs). Exploring the mechanism of action of key factors will provide reliable molecular markers for early diagnosis of aortic coarctation.

Screening of mRNA Related to Aortic Dissection.
Retrieval of mRNA expression data set related to aortic dissection in NCBI GEO database [14], download address is https:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc�GSE52093. e data contain gene expression profiling of dissected ascending aorta with that of control.

Differential mRNA Expression Related to Aortic
Dissection. For the downloaded expression spectrum data in the original CEL format, the R software (version 3.3.2) package oligo [15] was used to correct the expression value background and normalize the expression spectrum data, including the conversion of the original data format, the supplement of missing values, the background correction (MAS), and the data standardization by quantiles. e gene expression matrix was divided into the case group and control group. e significance of p value of gene expression difference was calculated by the nonpaired t-test provided by lima [16], and the p value was corrected by BH. e adjusted p value < 0.05 and |log2 (foldchange)| > 1 was considered as the screening threshold for differential expression genes (DEGs).

GO and KEGG Enrichment
Analysis. Gene Ontology (GO) enrichment analysis was performed by the BiNGO plugin of Cytoscape software v3.6.0 to understand the biological functions of DEGs [17]. Statistics were performed using hypergeometric tests with the Benjamini and Hochberg false discovery rate (FDR) correction, and the significance level of GO terms was defined as FDR ≤ 0.01. DAVID 6.8 (2019-12-03) [18] the functional annotation program was used to perform Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis with the following settings: gene count 5, easy 0.05.

Interaction Network and Expression Pattern Analysis.
Interaction of all the differential expression genes was analyzed using STRING database [19]. e obtained file of gene interactions was imported into Cytoscape software, for the visualization of gene interaction network.
e Cytoscape plugin, ClusterONE [20,21] was used for the subnetwork module analysis; genes in the significant enriched modules were further applied for functional enrichment analysis. e Cytoscape plugin, cytoHubba [22], was used to explore the hub genes from the gene interaction network, and the genes with degree value larger than 5 were considered as hub genes.

Cell
2.5.1. Cell Culture. Human aortic smooth muscle cells lines (HAoSMCs) were purchased from PromoCell (Heidelberg, Germany). All cell lines were cultured in a smooth muscle cell growth medium (PromoCell) and maintained in a humidified atmosphere containing 5% CO 2 at 37°C.

Apoptosis Assessment of Aortic Smooth Muscle Cells.
Cell apoptosis was detected using an Annexin V-FITC Apoptosis Detection Kit (Biomiga, China) according to the manufacturer's protocol. After 72 hours of transfection, human aortic smooth muscle cells were digested by trypsin without EDTA, then harvested and washed three times with PBS. Cells were resuspended in an Annexin V binding buffer, labeled with the Annexin V-FITC and propidium iodide (PI) sequentially and incubated at room temperature for 5-10 min, protected from light. e flow cytometry system (BeamCyte 1026, BeamDiag, China) was performed at an excitation of 488 nm and an emission of 530 nm to detect the apoptotic cells. Caspase 3 is a key enzyme in the 2 Evidence-Based Complementary and Alternative Medicine process of apoptosis and the activity was determined using the Caspase 3 Activity Assay Kit (Beyotime, China).

Cell Proliferation Detection of Aortic Smooth Muscle
Cells. Cell proliferation was analyzed using the Cell Counting Kit-8 (Beyotime, China). At 72 hours after transfection, the CCK-8 solution was added into the medium. One hour later, the absorbance at 450 nm was measured, which has a linear relationship with the number of cells.
2.6. Analysis 2.6.1. qRT-PCR Analysis. Total RNA was isolated with the Trizol reagent (Invitrogen, America) and cDNA was synthesized with a SuperScript III Reverse Transcription kit (Invitrogen, America) following the manufacturer's protocol. qRT-PCR reactions with a 20 μL volume were prepared in triplicates by adding cDNA to the SYBR qPCR mix (Invitrogen, America) and run on an Applied biosystems (USA) detection system according to the manufacturer's instructions. Data were analyzed with StepOne Software. e primer pairs used for qRT-PCR are provided in Table S1. Relative gene expression levels were quantified using the 2 −ΔΔCt method [23]. ACTIN was used as the internal quantitative control.

Differential mRNA Expression Related to Aortic
Dissection. To identified genes associated with aortic dissection, we applied the "lima" R package to screen DEGs between normal tissue samples and aortic dissection samples. A total of 111 upregulated and 223 downregulated genes were obtained (Figure 1(a)). e results of the GO analysis showed that biological processes such as extracellular matrix organization, angiogenesis, and regulation of smooth muscle contraction were found to be significantly enriched among DEGs (Figure 1(b), Table S2). In the disease database, it was found that the differential genes were significantly enriched to hypertension, abdominal aortic aneurysm, connective tissue diseases, myocardial infarct, aortic aneurysm, vascular diseases, atherosclerosis, and cardiovascular diseases (Figure 1(c)). KEGG enrichment analysis showed that ECM-receptor interaction and vascular smooth muscle contraction were significantly enriched in DEGs (Figure 1(d)), suggesting that alterations of the ECM might be responsible for the malignant progress of aortic dissection.

Interaction Network and Expression Pattern Analysis.
Of the 334 DEGs related to aortic dissection, there are 314 pairs of interaction among 126 genes basing STRING database. We divide these interactions into 20 submodules using ClusterONE (Table S3). 126 genes were sequenced according to the strength of interaction, and the core genes were screened. Finally, we found that submodule 2 (p � 0.00000139215) was significantly related to the occurrence of aortic dissection. e degree value of SERPINE1 gene in submodule 2 is 5, and the interaction network diagram shows that the genes interacting with SERPINE1 gene include ACTN4, GAS6, TIMP1, SERPINA1, and PLAUR ( Figure 2, Table 1).

Effects of SERPINE1 Gene on Apoptosis and Proliferation of Human Aortic Smooth Muscle Cells.
To explore the function of SERPINE1 in apoptosis and proliferation of HAoSMC, we transfected in vitro cultured normal HAoSMC with a SERPINE1-overexpressing adenovirus vector (Ad-SERPINE1) and SERPINE1 silencing lentivirus vectors (si-SERPINE1). e positive rate of SERPINE1 was 22.2% in normal aortic smooth muscle cells, 95.3% in Ad-SERPINE1 and 9.8% in si-SERPINE1 (Figure 3(a)). en, western blots and qRT-PCR were used to confirm efficacy of transfection. In HAoSMC transfected with Ad-SERPINE1, SERPINE1 mRNA and protein expression were 2.34-and 2.30-fold of the control group (Figure 3(b) and 3(c)). In HAoSMC transfected with si-SERPINE1, SERPINE1 mRNA and protein expression were 0.5-and 0.3-fold of the control group (Figures 3(b) and 3(c)). In addition, cell apoptosis rates of HAoSMC were detected using flow cytometry and it increased obviously to 23.32% in Ad-SERPINE1 (Figure 4(a)). Caspase 3 is a key signaling molecule in the process of apoptosis and Bcl-2 is an antiapoptotic and can inhibit apoptosis. Western blot showed that SERPINE1 overexpression resulted in an upregulated expression of caspase 3 (1.89-fold of control) and a downregulated expression of Bcl-2 (0.44-fold of control), which was consistent with the result of caspase 3 activity analysis (Figures 4(b) and 4(c)). e effect of SERPINE1 overexpression on cell proliferation was evaluated with CCK8 assay. e results showed that the proliferation ability of HAoSMC transfected with Ad-SERPINE1 was increased at 72 hours, while that of HAoSMC transfected with si-SERPINE1 decreased ( Figure 5).

Effects of the SERPINE1 Overexpression on Gene Expression Interacted with It.
In the above, we have screened 5 DEGs that predicted to have interacted with SERPINE1, including ACTN4, GAS6, TIMP1, SERPINA1, and PLAUR. e SERBP1 gene can bind to the 3′-most 134 nt of the SERPINE1 mRNA to regulate of mRNA stability. erefore, mRNA expression levels of these six genes were also detected after HAoSMC transfection with SERPINE1 genes. In the Ad-SERPINE1 groups, the expression of SERBP1, TIMP1, PLAUR, and SERPINA1 was upregulated and that of ACTN4, GAS6 was downregulated. However, in the si-SERPINE1 group, the opposite results were observed ( Figure 6).

Discussion
Studies have shown that AD may be a disease involving multiple systems and organs, with a high incidence of complications. When AD occurs, patients suffer from impaired renal function, decreased creatinine clearance rate, decreased pulmonary function, syncope, cognitive function, and other neurological symptoms [34,35]. In addition, some AD tears involve the coronary arteries, causing changes in troponin and electrocardiogram, making early and rapid diagnosis difficult [36]. e initial assessment and diagnosis of AD by routine examination is not easy at present, the misdiagnosis rate is high, and routine laboratory tests have no specific significance for the diagnosis of aortic coarctation and can only be used to exclude other diagnostic possibilities [37]. Occasionally, the acute onset of aortic coarctation may be associated with stress-related leukocytosis or anemia due to severe bleeding and massive blood flow into the false lumen, with individual diffuse intravascular coagulation [38]. Serum aminotransferases are generally not elevated unless myocardial infarction occurs with involvement of the coronary artery by a clotted hematoma [39]. Serum amylase has been reported to be increased when plasma cavity hematoma is present, or when the superior mesenteric artery is involved and involves the pancreas [40,41]. Hematuria has also been reported in the presence of renal involvement, and hemorrhagic cerebrospinal fluid is reported in the presence of stroke [42,43]. Once the dissection ruptures, the patient may die of massive hemorrhage within a few hours. erefore, it is extremely important to find an early and convenient diagnostic method that is suitable for AD   Evidence-Based Complementary and Alternative Medicine patients clinically [44]. In recent years, markers of vascular smooth muscle cell injury have focused on smooth muscle myosin heavy chain, creatine kinase isoenzyme BB. However, these biomarkers do not have a wide range of high sensitivity and specificity. To screen for biomarkers that can be used for early diagnosis of AD, we used a bioinformatics approach to analyze the expression profiles of dissected ascending aorta with control genes from the GEO database, which provides important information for screening biomarkers of ascending aorta. e molecular pathogenesis of aortic dissection is considered mainly with the structure and function of the ascending aorta vascular middle smooth muscle cells change, including smooth muscle cell proliferation, migration, collagen and elastic fiber ratio, leading to the loss of the ascending aorta artery elasticity, formed in the hypertension induced by aortic dissection. Vascular smooth muscle cells are the most important cells in the middle layer of ascending aorta. Smooth muscle cells are usually in a contractile state, that is, mature and differentiated smooth muscle cells with a contractile function. However, long-term exposure to external stimuli causes it to undergo phenotypic changes. e other form of smooth muscle is the proliferative type, which does not have the contraction function but has strong proliferation and migration activity. Kimura et al. found that several genes related with vascular smooth muscle contraction are significantly differentially expressed in dissected ascending aorta by microarray analysis [45]. Several studies have shown that the pathogenesis of aortic coarctation is related to the phenotypic transformation of vascular smooth muscle cells. Wei et al. (2017) found that downregulation of the talin-1 expression induced proliferation and migration of vascular smooth muscle cells in aortic coarctation [46].  revealed that EZH2 can inhibit autophagy of vascular smooth muscle cells and affect the pathological process of aortic coarctation [47]. Iida et al. (2018) showed that the overexpression of PCSK9 in vascular smooth muscle cells may promote the pathological process of aortic coarctation [48].
us, our analysis of gene expression profiles revealed that differentially expressed genes were enriched in the vascular smooth muscle contractile pathway, which is correct and consistent with previous studies. MYH11 is a smooth muscle cell-specific contractile protein that causes phenotypic transformation of smooth  Evidence-Based Complementary and Alternative Medicine muscle cells [49]. CNN1 is a contractile regulatory protein expressed by VSMC, mainly in contractile cells, and is an important marker of phenotypic transformation in VCMC cells [50].
SERPINE1 is the key gene related to aortic dissection obtained through interaction network analysis of differentially expressed genes. It has been reported that SERPINE1 is highly expressed in tumor tissues of patients with various types of cancer and is involved in cancer progression. Furthermore, the SERPINE1 overexpression plays an important role in acute respiratory distress syndrome (ARDS) due to corona virus 2019-associated coagulation disorder, suggesting the therapeutic potential of targeting fibrinogen activator inhibitor-1 in corona virus 2019 [51]. Its biological roles in tumors include inducing angiogenesis, promoting cell invasion and migration, maintaining proliferation, and resisting apoptosis. Moreover, it has also been used as a potential biomarker for a variety of cancers in recent years [52][53][54][55]. Meanwhile, Kimura et al. found that the expression level of SERPINE1 in acute type A aortic dissection (ATAAD) patients was upregulated [45]. Our research results also showed that the expression level of SERPINE1 in aortic dissection was 2.97 times higher than that in normal tissue. It is speculated that there is a relationship between the expression of SERPINE1 gene and the occurrence of aortic dissection. In order to further clarify the role of this gene in aortic dissection, we examined the effects of SER-PINE1 overexpression and expression inhibition on human aortic smooth muscle cells apoptosis and proliferation in vitro. e results showed that SERPINE1 could promote human  Evidence-Based Complementary and Alternative Medicine aortic smooth muscle cells apoptosis and proliferation simultaneously. Moreover, 5 DEGs were predicted to have interacted with SERPINE1 by STRING database analysis, including ACTN4, GAS6, TIMP1, SERPINA1, and PLAUR. e qRT-PCR results showed that Gas6 and ACTN4 were negatively correlated with the SERPINE1 expression. Melaragno reported that Gas6 could activate the PI3K/Akt pathway by increasing the expression of phosphorylated Akt and inhibit the apoptosis of vascular smooth muscle cells [56]. Qiu illuminated that Gas6 was critical in VK2-mediated functions that attenuate CaCl-and β-GP-induced VSMC calcification by blocking apoptosis [57]. It is therefore hypothesized that the Gas6 gene may be a key factor in regulating apoptosis of vascular smooth muscle cells in aortic coarctation. TIMP1 is a tissue matrix metalloproteinase inhibitor that inhibits the biological activity of MMPs. e concentrations of MMP8, MMP9, TIMP1, and TIMP2 in patients with aortic coarctation have been reported to be higher than those in normal tissues, but MMP8 and MMP9 are much higher than TIMP1 and TIMP2, resulting in aortic coarctation disequilibrium. TIMPs/MMPs ratio, enhanced protein hydrolytic activity, accelerated extracellular matrix degradation, and aortic coarctation occur [58].
In traditional Chinese medicine, the etiology of aortic dissection is a kind of disease caused by congenital insufficiency, acquired dystrophy, and internal invasion of exogenous pathogens, resulting in deficiency of yin and blood, which cannot nourish the heart and weakens the heart [59]. Generally, the methods of nourishing blood and nourishing qi, nourishing the heart and veins, and treating according to syndrome are used to deal with it. Chinese medicine treatment also includes acupuncture rehabilitation, traditional Chinese medicine ingredients, etc., and also symptomatic treatment according to individual physique. Conservative drug therapy reduces the patient's blood pressure to the lowest value that can be tolerated by antihypertensive drugs [59]. In addition, the patient's heart rate can be controlled as much as possible by oral medication to reduce the stress on the heart [60]. But conservative treatment also has risks. Once a patient's aortic dissection ruptures, the patient can die at any time. From this point of view, aortic dissection can not be improved only by Chinese medicine treatment. Only complete closure of the dissection can be cured, so aortic dissection requires timely surgical treatment.
In summary, through bioinformatics analysis of the sequence data of mRNA related to aortic dissection in GEO database, the gene expression regulatory network of aortic dissection was constructed. rough the screening of key node genes, the SERPINE1 gene in submodule 2 was significantly related to aortic dissection. e functional analysis in human aortic smooth muscle cells (HAoSMCs) showed that SERPINE1 may be involved in the pathophysiological process of aortic dissection by promoting apoptosis and proliferation.
e comprehensive analysis confirmed that Ad-SERPINE1 si-SERPINE1 Control Figure 6: Reverse transcription-quantitative PCR was performed to detect the expression of SERBP1, TIMP1, PLAUR, SERPINA1, ACTN4, and GAS6 in aortic smooth muscle cells after transfection SERPINE1 gene. 8 Evidence-Based Complementary and Alternative Medicine the SERPINE1 gene is a reliable molecular marker for early diagnosis of aortic coarctation, and based on the molecular mechanism, we speculate that possible new drugs targeting the SERPINE1 gene are possible in the future. But there is still a long way to go to develop drugs and treatments targeting the SERPINE1 gene. Before that, it is very necessary to find an effective prognostic method to reduce various risks during and even after surgery, which may become the future research direction for a period of time.

Data Availability
All datasets used and/or generated during the present study are available from the corresponding author on reasonable request.

Disclosure
Dong Li and Cang-Song Xiao are the co-first authors.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Dong Li and Cang-Song Xiao contributed equally to this work. Table S1. Primers used in qRT-PCR.